21 TRENDS IN PREP ARATIVE HPLC Ernst Kuesters 21.1 INTRODUCTION Directly from its beginning—now 100 years ago, when Michail Tswett devel- oped the principles [1, 2] with the isolation of chlorophyll—chromatography has always been a preparative technology, and its value in producing com- pounds of high purity cannot be overemphasized. It was Paul Karrer [3] who stated very early “. . . it would be a mistake to believe that a preparation puri- fied by crystallization should be purer than one obtained from chromatographic analysis. In all recent investigations chromatographic purification widely sur- passed that of crystallization.” and Leslie Ettre, although not distinguishing between analytical and preparative separations, denoted chromatography as “the separation technique of the 20th century” [4]. From a historical point of view, the beginnings of preparative isolation of natural compounds were cum- bersome. For example, it is reported [5] that six years of work and processing of 30 tons of strawberries was needed to finally obtain 35mL of an oil, the essence of the fruit. This situation changed dramatically in the 1960s with the theoretical understanding of the chromatographic process, the development of high-performance liquid chromatography, and the synthesis of highly selec- tive stationary phases. As a result of these improvements, the isolation of natural compounds with preparative chromatography on production scale (e.g., drug substances from fermentation processes) is still state of the art,even after 100 years. Today, preparative HPLC has also become a powerful technology in phar- maceutical development and production either for isolation of impurities, for 937 HPLC for Pharmaceutical Scientists, Edited by Yuri Kazakevich and Rosario LoBrutto Copyright © 2007 by John Wiley & Sons, Inc. chromatographic purifications, or as part of a scale-up process and subse- quently has been reviewed in a lot of monographs [6–10]. The term prepara- tive amount thus covers the range from milligram quantities (amounts for structure elucidation, analytical characterization, toxicology, or reference material) to large-scale production of tons of intermediates and drug sub- stances. The separations therefore can be performed on all types of columns, starting from analytical ones up to production scale columns with 1-m i.d and several meters in length. Typical applications are summarized in Table 21-1. The success of preparative HPLC on a production scale has been made pos- sible because of significant improvements made in several areas like (i) column technology (today, mainly compressed columns are used), (ii) packing mate- rials (pressure stable spherical particles with high homogeneity, either non- chiral or chiral), and (iii) the understanding of the nonlinear process in preparative HPLC (overloaded conditions) which resulted in new methods to determine the adsorption isotherms and which consequently led to new con- cepts like displacement chromatography and simulated moving bed (SMB) chromatography, where the knowledge of such adsorption isotherms is a pre- requisite for the design of the corresponding separation process. The aim of this chapter is to highlight current developments in these various fields of preparative HPLC, with particular emphasis on applications that have been developed at Chemical & Analytical Development at Novartis Pharma AG. Drug substance purifications from biological and synthetic sources are presented, along with the separation of chiral and/or achiral molecules on chiral stationary phases and typical isolations of by-products. Special attention is given to the determination of adsorption isotherms and their interplay with respect to the layout of chromatographic processes as well as the choice of 938 TRENDS IN PREPARATIVE HPLC TABLE 21-1. Order of Magnitude and Purpose of Purified Amounts Obtained from Preparative Chromatography Amount of Stationary Amount of Column Type I.D. (mm) Purpose Phase (g) Product (g) Analytical 1–5 Isolation of reference 0.2–3 0.0002–0.003 substances (MS or NMR) Analytical— 5–10 Starting materials 0.003–25 0.003–0.1 semipreparative for toxicology Semipreparative 10–40 Intermediates for 25–100 0.1–5 —preparative lab synthesis Pilot plant 100–300 Manufacturing of 100–1000 20–5000 drug substances for pharmaceutical development Production 300–1,500 Manufacturing of 1,000–4,000,000 kg-tons trade products technology. The applications have been selected in such a way that a broad variety of technologies like multiple injection, recycling, displacement, and SMB chromatography is covered. On-line detection tools have to fulfill other demands in preparative chromatography than in analytical chromatography. A special section has been devoted to this aspect below, and an instrument that was developed in-house is presented. 21.2 METHOD DEVELOPMENT IN PREPARATIVE HPLC Since chromatography scales up linearly and independently from the selected technology (rationales when making a choice will be given later on), the column containing the stationary phase is still the heart of the system. Method development will therefore always start with the selection of the best station- ary and mobile-phase composition to achieve an optimum in productivity, which does not necessarily mean an optimum in selectivity.For example, a high selectivity of α>10 has been obtained for the enantiomeric separation of β- blocking agents like pindolol using amylose- or cellulose-derived stationary phases, but the poor solubility of the racemates in the mobile phase (hexane/2- propanol mixtures) will never result in an economic separation process. This situation can be significantly improved by (i) solvent switch and (ii) adding of bases or acids, which leads to higher solubility and productivity, although the selectivity decreases. Figure 21-1 shows the separation of the enantiomers of pindolol under different conditions [11, 12]. Even though the addition of TFA clearly results in very distorted isotherms, the situation from the point of view of the preparative separation is much improved, with the throughput increas- ing from 322 to 860g of racemate per kilogram of chiral stationary phase per day. Nevertheless, as a rule of thumb, in most cases higher productivities have METHOD DEVELOPMENT IN PREPARATIVE HPLC 939 Figure 21-1. T he effect of mobile-phase additives on pindolol on Chiralcel-OD (analytical column). Mobile phase: (a) Methanol/diethylamine = 99.9/0.1, 20°C. (b) Hexane/ethanol/trifluoroacetic acid = 60/40/0.5, 40°C. (c) Conditions as for (b), but 25-mg load. (Reprint from reference 12, with permission.) been obtained under separation conditions where high selectivities have been identified. Therefore, in parallel, parameters like solubility of the sample in the mobile phase, capacity of the stationary phase, stability, and work-up of product containing fractions have to be determined. Once a robust system has been developed, the possibilities of scale-up (solubility of sample, stability of product in mobile phase, work-up, etc.) are investigated in the next step. And finally the adsorption isotherms are measured as a guide to the appropriate and economic technical realization on pilot plant or production scale. 21.2.1 Optimization of Selectivity The first step, the search for an appropriate chromatographic system, can be explored with the aid of analytical columns or even more easily in the case of straight-phase chromatography with thin-layer chromatography (TLC). In the case of chiral separations with chiral stationary phases (CSP), a quick survey of separation strategies is provided by using electronic databases like Chir- base in advance. Since each type of column overloading will result in a loss of separation, the method development should start with the search for a suffi- cient peak resolution R s . Under analytical conditions, the peak resolution R s is the result of the interplay of selectivity or separation factor α, retention time, and column performance according to equation (21-1): (21-1) where a is the separation factor (selectivity) = k 2 /k 1 for k 2 > k 1 ; k 1 and k 2 are the capacity factors of substance 1 and 2, respectively; and N is the plate number. A rough estimation nicely highlights the contribution and importance of a well-developed separation factor. Whereas changes in k from 3 to 5 only improve the peak resolution by 10.7% and a doubling of N by 41.4%, the increase of selectivity from 1.2 to 2.2 will result in an improvement of 83.3%. Since in most cases the technical parameters like particle size and pressure are given and used under optimum conditions, the search for high selectivity cannot be overemphasized. The main parameters to optimize the separation factor and peak resolu- tion, respectively, are as follows: • Appropriate stationary phase (which not only seeks for the appropriate polarity of the material; the “same stationary phase” from different sup- plier may have a significant influence on the selectivity because of differ- ences in the manufacturing process). • Appropriate mobile phase (which includes the choice and composition of solvents, additives, and pH value). R k k N S =− () + () 1 4 1 1 a 940 TRENDS IN PREPARATIVE HPLC • T emperature. Especially the latter parameter should not be underesti- mated. Although, as a rule of thumb, achiral separations are often per- formed at elevated temperatures, it is generally believed that separations on chiral stationary phases should best be performed at lower tempera- tures. Nevertheless, sometimes it turns out that chiral separations are entropy controlled and better selectivities are obtained at higher temperatures [13–16]. Once the right set of parameters has been identified, computer-aided opti- mization using modified sequential simplex or central composite design methods can be applied to further fine-tune the separation under investiga- tion, as has been published for the optimization of reverse-phase HPLC [17–20] and chiral separations [21–23]. 21.2.2 Scale-Up of Analytical Methods 21.2.2.1 Overloading. The fundamental difference between preparative chromatography and analytical chromatography is the sample amount being injected. In analytical chromatography the sample amount is extremely small with regard to the amount of stationary phase (<1:10,000) and the chro- matography is consequently performed in the linear range of the adsorption isotherms of the components being separated. A rough calculation at that point nicely demonstrates that a simple linear enlargement will never provide an economic process. Therefore the injection amount will successively be increased, which in the first instance will result in an adequate increase of peak heights and peak areas while leaving the retention times and separation factors unaffected.A further increase of the sample amount then will result in an over- loading of the column and in deformed and moving peaks as a consequence of a shift in the nonlinear range of the adsorption isotherms. Concave isotherms will provide broader tailing peaks with shorter retention times, whereas convex isotherms will show broader fronting peaks with greater retention times. The separation of course will become poorer; nevertheless, as long as it is sufficient, the process will become more and more economic. The increase of the injected quantity until the two peaks touch is called touching- band optimization [24], and an example is given in Figure 21-2 for the sepa- ration of an artificial mixture of epothilone A and B. This optimization approach has the advantage of being fast and simple, but it often overlooks specific effects that happen at larger loads. These effects concern the displacement of one product by another and have been described by Guiochon and co-workers [25–28] and Cox and co-workers [29–31]. The interplay of adsorption isotherm, peak form, and capacity factor k during overloading of a column is depicted in Figure 21-3 [32]. Sometimes, during the course of determining the capacity of the stationary phase and the adsorption isotherms, it turns out that significant preparative amounts of reference material can easily be obtained even with analytical METHOD DEVELOPMENT IN PREPARATIVE HPLC 941 942 TRENDS IN PREP ARATIVE HPLC Figure 21-2. Separation of 247mg of epothilone A (first eluting) and B (structure given below) on a semipreparative reversed-phase ODS column (25-cm × 2.0-cm i.d.). Particle size 11µm, mobile phase acetonitrile/water = 4/6 (V/V), flow rate 15mL/min, UV detection 250nm. Figure 21-3. T he effect of adsorption isotherm on peak form and capacity factor k during overloading of a column. c s and c m = concentration of substance in the station- ary and mobile phase; A, B, C, D refer to substance A, B, C, D, respectively. columns. Given the good solubility of a racemic morphanthridine in the mobile phase and the large separation factor , the author decided to estimate the capacity of the CSP for the given separation [33]. The injection amount sys- tematically increased to estimate the final value for which a baseline sepa- ration could be observed. To obtain on-scale peaks, UV detection was carried out at 290nm, and the automatic injection device was replaced by a manual loop with different volume sizes.After several runs the endpoint was the injec- tion of 100mg of racemate dissolved in 250 µL of hexane/2-propanol = 1/1 (V/V). The preparative chromatogram of this run is shown in Figure 21-4. It is obvious from the individual peak shapes that both enantiomers follow dif- ferent adsorption isotherms. Whereas for the first eluting enantiomer, a linear adsorption isotherm is observed, the corresponding one for the second eluting enantiomer is much more complex. Nevertheless, both enantiomers are sepa- rated to baseline and completely eluted within 15min. It is therefore obvious that even without further optimization, a daily yield of 9.6g of resolved race- mate can be achieved using an automatically injection device with repetitive injection. Based on this result, several interesting production scenarios can be derived. Just by increasing the inner diameter of the column, the production of ton amounts/year with a daily mobile phase consumption of less than 1m 3 may be easily achieved.The results of the calculations are summarized in Table 21-2. As can be taken from Table 21-2, a respectable amount of 96kg of race- mate can be resolved per day on a column containing 30kg of CSP. In a typical pilot plant environment, such a column belongs to the smaller ones and also METHOD DEVELOPMENT IN PREPARATIVE HPLC 943 Figure 21-4. Preparative enantioseparation of a morphanthridine analogue on an ana- lytical Chiralpak-AD column (250-cm × 4.6-mm i.d.). Mobile phase Hexane/2-propanol = 85/15 (V/V), 0.5 mL/min; temperature 40°C, UV detection 290nm, injection amount 100mg/250 µL hexane/2-propanol = 1/1 (V/V). (Reprint from reference 33, with permission.) the daily mobile-phase consumption of 7.2m 3 is not a technical hurdle .A fully automated chromatographic system would consequently provide a yearly pro- duction of 35 tons of resolved racemate. Later on (Section 21.4.4) it is shown that in most cases where conventional batch elution chromatography is com- pared with simulated moving bed (SMB) applications with the same amount of CSP, productivity can double and solvent savings up to 80–90% are achieved.Assuming such a production scenario for the above-mentioned mor- phanthridine analogue, a daily production of 192kg (corresponding to 70 tons/year) reflects a feasible order of magnitude. In addition, a daily solvent consumption of 720L is negligible from a production point of view. 21.2.2.2 Solubility and Self-Displacement. In the previous scenario, the feed concentration was gradually increased. This kind of overloading, called concentration overloading, comes to an end when the solubility product of the solute is achieved. A further increase of sample amount can then only be achieved with volume overloading, the injection of larger feed volumes into the column. Very often in practice the combination of both types of over- loading comes into operation. In the case of an excellent selectivity in combi- nation with a poor sample solubility, the addition of a more polar solvent to the feed solution may help to achieve a higher productivity. As a result of the slightly modified chromatographic system, a partial self-displacement is observed, visualized by a doubling of the eluting peaks. Since, in addition, the retention is shifted to shorter retention times, this improvement will also come to an end when the first compound leaves the column unretained with t 0 . Therefore sometimes the reverse occurs—for example, when a good sample solubility meets excellent elution conditions. To avoid peak elution during the injection period, the polarity of the feed solution is changed by addition of a 944 TRENDS IN PREPARATIVE HPLC TABLE 21-2. Calculated Production Scenarios for a Preparative Enantioseparation of a Morphanthridine Analogue on Chiralpak-AD Analytical Pilot Plant Production Amount of CSP Column Column Column 3g 3kg 30kg Batch Elution Mode Resolved racemate/day 9.6g 9.6kg 96.0kg Resolved racemate/year 3.5kg 3.5 tons 35.0 tons Solvent consumption/day 0.72L 0.72m 3 7.2m 3 SMB Mode Resolved racemate/day NA 19.2kg 192.0kg Resolved racemate/year NA 7.0 tons 70.0 tons Solvent consumption/day NA 72L 0.72m 3 NA, not applicable with respect to preparative method. further solvent in such a way that the solubility of the feed solution decreases and takes significantly larger injection volumes into account. Injection times of 30min. and longer are acceptable as long as the sample stays retained at the top of the column. After the injection is finished, the solutes are eluted with the mobile phase that has a better solubility.An example of this approach has recently been published for the purification of discodermolide [34] (Figure 21-4). A 38-g sample of crude product (82.4%) was dissolved in 11.2L of 2-propanol and diluted with 78.4L of water. After injection of this feed solu- tion onto a column containing 15kg of ODS-RP-18 reversed-phase phase silica gel, the drug substance was eluted with a mixture of acetonitrile/water = 25/75 (V/V) in an isocratic mode. It is noteworthy that in the large-scale synthesis of 60g discodermolide, 39 steps (26 steps in the longest linear sequence) and several chromatographic purifications were involved. A chromatographic purification of such a “small” amount of a highly active drug substance which delivered sufficient material for early-stage human clinical trials is the method of choice, since extremely pure material is obtained on pilot plant equipment in a very short time. Figure 21-5 shows a semipreparative purification of dis- codermolide during method development on a lab-scale column and highlights the effectiveness of the purification step. 21.2.2.3 Purity of Solvents, Stability of Products and Work-up. The quality aspect of the solvents used as mobile phases should not be forgotten, since the evaporation residue from the mobile phase can be significant. Assuming an average product concentration of 1–2g/L mobile phase, it becomes obvious that an evaporation residue of 10mg/L solvent leads to 1 g of evaporation METHOD DEVELOPMENT IN PREPARATIVE HPLC 945 Figure 21-5. Purification of 101mg of crude discodermolide on 46 g of YMC-OD-A 5–15µm (column: 250-mm × 20-mm i.d.). The drug substance is dissolved in 31.4 mL of 2-propanol and 220.6mL of water are added. The feed solution is pumped with a flow rate of 10mL/min onto the column, and the compounds are eluted afterwards with a mixture of acetonitrile/water = 2/1 (V/V), flow rate 15 ml/min; UV detection 220 nm. residue in 100g of product. Solvents that are used in preparative chromatog- raphy should therefore have an evaporation residue of <10 −4 g/L. To ensure a good quality of the product, it is therefore sometimes necessary to purify the solvents in advance prior to their use as mobile phase. This not only will have an influence on the product quality, but also may, in addition, by removing heavy metals and/or stabilizers, have an impact on the resolution and there- fore also affect the ruggedness of the chromatographic process. As has been shown by Dingenen [35], the switch from one supplier to another can lead to the complete loss of selectivity in a chromatographic step. Once a chromatographic system has been identified for a preparative purpose, the stability and work-up procedure of the product-containing frac- tions should be investigated. Sometimes it turns out that the products cannot be isolated by simple removal of the solvents, because of thermal instability or too basic or acidic conditions in the mobile phase. In such a case an appro- priate extraction procedure from the mobile phase may help to isolate the products. 21.2.3 Adsorption Isotherms and Their Determination The most common technique used in preparative chromatography is still iso- cratic batch elution. However, more sophisticated technologies like recycling, gradient elution, displacement, or the simulated moving bed (SMB) process are being increasingly applied to enhance productivity and yields. A fair com- parison between these rivaling technologies is only possible on the basis of real occurring concentration profiles that agree excellently with the theoreti- cal predictions. The substantial progress that has been achieved in modeling preparative chromatography was reviewed recently [36–38]. The underlying equilibrium-dispersion model, for which the mass balance for solute i in a N component mixture and a volume element is given in equa- tion (21-2), has been very often successfully applied to quantify chromato- graphic processes under overloaded conditions. (21-2) In this equation, c is the concentration in the fluid phase and q is the quantity in the solid phase. The column porosity e (expressed as phase ratio F = (1 − ε)/ε) defines the fraction of the fluid phase in the column. Furthermore, u stands for the linear velocity and t and x are the time and space coordinates, respectively. All contributions leading to band-broadening are lumped in a simplifying manner into an apparent dispersion coefficient, D ap . In equation (21-2), it is assumed that the two phases are constantly in equilibrium expressed by the adsorption isotherms. Due to the nonlinear character of the isotherm equations, the solution of equation (21-2) requires the use of numeri- ∂ ∂ ∂ ∂ ∂ ∂ ∂ ∂ c t F qc t u c x D c x iNcccc i i i ap i N i + () += = = () 2 2 12 1, , , with , , , 946 TRENDS IN PREPARATIVE HPLC [...]... 45% (Figure 21 -21), an overall yield for the whole process of 81% was achieved with a final purity of >98% Figure 21-22 illustrates the internal concentration profiles for AD and 9-epi-AD along the columns of the SMB unit obtained for the chosen operating conditions Technical details can be taken from the literature [127] Example 3 Enantiomeric Separation of DOLE The interest in SMB for performing large-scale... Phases The most widely used packing materials in preparative HPLC are the silicabased particles Although irregular particles are still available, for preparative columns most applications tend to use spherical packings, since better packings are obtained and for additional reasons mentioned below Underivatized silica and C18 reversed-phase material (for most applications) are available in packed column as... being used in preparative HPLC, and the interested reader is referred to the literature [53] Nevertheless, two types of stationary phases have emerged during the last years which seem to be cornerstones of new innovations Their importance is still increasing and they are therefore discussed in a little bit more detail: 952 TRENDS IN PREPARATIVE HPLC • Chiral stationary phases for the separation of chiral... enantiomers by preparative HPLC is now widely used, and a large number of CSP are commercially available As a method to produce both enantiomers of a drug candidate directly at the beginning of the clinical development, it is becoming more and more attractive because it allows the rapid and easy supply of amounts for biological testing, for toxicological studies, and even, in a later stage, for clinical testing... profiles of the individual enantiomers are meanwhile systematically required by health authorities for new drugs submitted for registration In addition, the concurrent development of simulated moving bed chromatography (a chromatographic system that ideally separates two component mixtures, see later) was fortunate for the boom in enantiomeric separations now reaching a production scale Several reviews have... columns should avoid the formation of such voids This hurdle can be overcome by using compression techniques This does not mean that the redistribution will not happen, but the consequences are eliminated Several compression methods have meanwhile been described in the literature [64] and are used for preparative HPLC Nevertheless, it should be pointed out that most applications are performed with equipment... excellent designed countercurrent process Nevertheless, this comparison will become more favorable for batch elution when the full capacity of the column is being used It should not be forgotten for preparative runs in isocratic mode that the process can be optimized in such a way that several separations can be performed successively on a column until the compounds of the first injection elute The net elution... cost of the displacement method: enough material for about 14 displacement runs can be generated from one concentration run on a column of the size used for displacement Moreover, preconcentration can be performed on a cheaper and coarser sorbent, if necessary 21.4.4 Simulated Moving Bed Chromatography Counter current processes have proven to be superior for the separation of binary mixtures in comparison... process on the shifting time tshift For the net flow ratios, equation (21-9) holds mj = ˙ Vj e − , ˙solid 1 − e V j = I, , IV (21-9) Most critical for a successful separation are the net flow ratios for the regions II and III upstream and downstream of the feed position, that is, mII and mIII Explicit equations are given to calculate regions in the mII, mIII plane for the constant selectivity Langmuir... suitable isotherm equation has to be chosen For mixtures, the model equations are usually coupled to take into 950 TRENDS IN PREPARATIVE HPLC account the competition for available adsorption sites The so-called multiLangmuir equation (21-7) was found to represent a lot of experimental data satisfactorily qi = ai Ci N 1 + ∑ bj C j , i = 1, , N (21-7) j =1 For enantiomeric separations, the modified competitive . preparative HPLC has also become a powerful technology in phar- maceutical development and production either for isolation of impurities, for 937 HPLC for Pharmaceutical. DEVELOPMENT IN PREPARATIVE HPLC 947 948 TRENDS IN PREP ARATIVE HPLC Figure 21-6. Experimental setup of ECP (a), MDM (b), and ADM (c) method for the determination